In the ReadyProbes Streptavidin/Biotin Blocking Solution (1X) protocol, why is there a step to add biotin to the sample?
The first step in this kit is to bind all endogenous biotinylated entities with an unlabeled streptavidin. The free D-biotin is added to bind to all biotin binding sites on the added streptavidin. The free biotin will be washed away before any other labels are added and all biotin binding sites will be occupied on the unlabeled streptavidin.
I am labeling cells with a biotinylated primary antibody, followed by a streptavidin dye conjugate, but I'm getting non-specific mitochondrial labeling, even with a streptavidin-only control. Why?
Mitochondria have endogenous biotin, which is being labeled by the streptavidin. You will need to block the endogenous biotin first, after you fix and permeabilize, but before you block for your primary antibody.
How is ReadyProbes Streptavidin/Biotin Blocking Solution (1X) different from other endogenous biotin blocking kits?
The only difference between the ReadyProbes Streptavidin/Biotin Blocking Solution (1X) and other endogenous biotin blocking kits is the use of streptavidin (as other kits use avidin) and the ease of use. In the ReadyProbes Streptavidin/Biotin Blocking Solution (1X), we provide the two components already pre-mixed and ready to use.